Transmitting tissue architecture in basal-relictual angiosperms: Implications for transmitting tissue origins

Carpel transmitting tissue is a major floral innovation that is essential for angiosperm success. It facilitates the rapid adhesion, hydration, and growth of the male gametophyte to the female gametophyte. As well, it functions as a molecular screen to promote male gametophytic competition and species-specific recognition and compatibility. Here, we characterize the transmitting tissue extracellular matrix (ECM) and pollen tube growth in basal-relictual angiosperms and test the hypothesis that a freely flowing ECM (wet stigma) was ancestral to a cuticle-bound ECM (dry stigma). We demonstrate that the most recent common ancestor of extant angiosperms produced an ECM that was structurally and functionally equivalent to a dry stigma. Dry stigmas are composed of a cuticle and primary wall that contains compounds that facilitate the adhesion and growth of the male gametophyte. These compounds include methyl-esterified homogalacturonans, arabinogalactan-proteins, and lipids. We propose that transmitting tissue evolved in concert with an increase in cuticle permeability that resulted from modifications in the biosynthesis and secretion of fatty acids needed for cuticle construction. Increased cuticle permeability exposed the male gametophyte to pre-existing molecules that enabled rapid male gametophyte adhesion, hydration, and growth as well as species-specific recognition and compatibility.     

Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbμ1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbμ1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbμ1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbμ1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.African trypanosomes (Trypanosoma brucei subspecies), the agents of African sleeping sickness, are alone among the kinetoplastid parasites (including Trypanosoma cruzi and Leishmania spp.) in having a pathogenic bloodstream stage that exists and replicates extracellularly in the mammalian host. This places unique constraints on the parasite in terms of dealing with host immune responses and on acquisition of essential nutrients. The parasite has evolved many strategies to deal with these constraints, the best known of which is the process of antigenic variation (9). Another is the lysosome, which impacts the host-pathogen balance in multiple ways. Trypanosomes have a single terminal lysosome that is the final repository of endocytic cargo acquired from the host serum for nutritional purposes (30), as well as for potentially lytic immune complexes removed from the cell surface (4, 8). Both endocytosis and lysosomal hydrolytic activities are differentially regulated through the trypanosome life cycle (11, 30), and there are stage-specific differences in the biosynthetic trafficking of essential lysosomal components (discussed below). The release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system (36). Finally, lysosomal physiology is critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species (38).Clearly, given its multiple roles in pathogenesis, biogenesis of the lysosome is critical to the success of trypanosomes as human parasites. As in all eukaryotes, lysosomal biogenesis is a balance between the proper sorting of newly synthesized membranes and proteins and recycling of established membranes and proteins internalized from the cell surface. In each case, protein sorting involves recognition of specific signals in cargo molecules by cellular machinery for inclusion in nascent transport vesicles destined for downstream delivery. Unique sets of cytoplasmic coat complexes at discrete intracellular locations serve the dual purpose of simultaneously mediating vesicle formation and selective cargo loading. The best characterized of these machineries is the clathrin/adaptin system for formation of coated vesicles at the Golgi apparatus and the plasma membrane (10, 41). Adaptor complexes (APs) are cytosolic heterotetramers that interact with specific signals in the cytoplasmic domains of membrane cargo proteins, such as dileucine motifs ([E/D]XXXL[L/I]) and tyrosine motifs (YXXØ, where Ø is a bulky hydrophobic residue). The prototypic AP complexes are AP-1 and AP-2, which function at the trans-Golgi network and plasma membrane, respectively. Both are composed of two large subunits (γ/β1 in AP-1; α/β2 in AP-2) and two smaller subunits (σ1/μ1 in AP-1; σ2/μ2 in AP-2). YXXØ motifs interact with μ adaptins, and dileucine motifs interact with combinations of adaptin subunits in both AP-1 and AP-2 (26, 40, 42). It is the large subunits, particularly β adaptin, that mediate clathrin recruitment (19, 44). Other APs, AP-3 and AP-4, with discrete subunit compositions, also exist. AP-3 functions in trafficking to lysosome-related organelles, such as melanosomes, and AP-4 may be involved in basolateral trafficking in polarized epithelial cells (10). The genome of the African trypanosome, T. brucei, encodes a complete complement of orthologous subunits for AP-1, AP-3, and AP-4 but has no genes for AP-2, the major adaptor complex mediating endocytosis in vertebrate cells (16). This is likely due to evolutionary loss, since the closely related T. cruzi has orthologues of all four APs.Two major lysosomal cargo proteins have been studied in T. brucei, the LAMP (lysosome-associated membrane protein)-like protein p67 and the cathepsin L orthologue trypanopain. p67 is a type I membrane protein with a large glycosylated lumenal domain and a short cytoplasmic domain (1, 27). In procyclic insect stage (PCF) trypanosomes, the cytoplasmic domain is both necessary and sufficient for lysosomal targeting of a heterologous reporter, and its deletion results in mistargeting of p67 to the cell surface (1). The cytoplasmic domain contains two canonical dileucine motifs, mutation of which also results in delivery to the cell surface (47). These findings strongly indicate the existence of cognate cytoplasmic machinery for lysosomal delivery of p67 in PCF trypanosomes. Strikingly, however, the cytoplasmic domain, and its motifs, are totally dispensable for lysosomal targeting in bloodstream stage (BSF) trypanosomes (1). Deletion of the cytoplasmic domain results in minor mislocalization to the cell surface, but p67 is still overwhelmingly delivered to the lysosome. Ongoing lysosomal targeting cannot easily be attributed to misfolding of the lumenal domain, as suggested by others (3), since the normal transport-associated patterns of p67 glycosylation and cleavage prevail in these deletion constructs.Less is known about targeting of soluble trypanopain. In mammalian cells, soluble hydrolases are targeted to the lysosome by the addition of mannose-6-phosphate (M6P) moieties in the Golgi apparatus, which serve as ligands for recognition and lysosomal targeting by downstream M6P receptors (28). Soluble hydrolases can also be sorted by receptors that recognize polypeptide motifs, such as sortilins in mammalian cells (12) and Vps10 in yeast (13, 32). These receptors have lumenal cargo recognition domains and cytoplasmic domains containing signals for late endosomal targeting and recycling. M6P-modified N-linked glycans are not found in trypanosomes, and genes encoding the necessary enzymatic activities are absent from the genome (16), ruling out this possibility for trypanopain sorting. However, the T. cruzi orthologue, cruzipain, has been shown to rely on peptide motifs in the N-terminal prodomain for targeting (24), raising the possibility of a sortilin/Vps10p-like sorting receptor. Although there are no obvious orthologues of these proteins in the T. brucei genome, overexpression of trypanopain in PCF trypanosomes leads to secretion, an observation that is consistent with saturation of a specific sorting receptor (S. S. Sutterwala and J. D. Bangs, unpublished observations).Having previously studied the innate signals involved in p67 targeting (1, 47), we now turned our attention to the cognate machinery for post-Golgi apparatus sorting. Specifically, we investigate the role of trypanosomal AP-1 in stage-specific biosynthetic trafficking to the lysosome using RNA interference (RNAi)-mediated silencing of the Tbμ1 (geneDB no. Tb927.7.3180 [www.genedb.org]) subunit as our primary strategy. Our results demonstrate that AP-1 and clathrin are critical for lysosomal targeting of p67 and trypanopain in PCF trypanosomes but that they are essentially dispensable in BSF parasites. These data, in conjunction with the behavior of p67-targeting mutants (1) and other trypanosomal secretory reporters, lead us to propose a simple model for stage-specific default trafficking in African trypanosomes. Although in some respects our results are similar to those of a recent publication using RNAi silencing of the Tbγ1 subunit of AP-1 (3), they differ in key aspects, leading us to significantly different conclusions.     

Metnase/SETMAR: a domesticated primate transposase that enhances DNA repair,replication, and decatenation

Metnase is a fusion gene comprising a SET histone methyl transferase domain and a transposase domain derived from the Mariner transposase. This fusion gene appeared first in anthropoid primates. Because of its biochemical activities, both histone (protein) methylase and endonuclease, we termed the protein Metnase (also called SETMAR). Metnase methylates histone H3 lysine 36 (H3K36), improves the integration of foreign DNA, and enhances DNA double-strand break (DSB) repair by the non-homologous end joining (NHEJ) pathway, potentially dependent on its interaction with DNA Ligase IV. Metnase interacts with PCNA and enhances replication fork restart after stalling. Metnase also interacts with and stimulates TopoIIα-dependent chromosome decatenation and regulates cellular sensitivity to topoisomerase inhibitors used as cancer chemotherapeutics. Metnase has DNA nicking and endonuclease activity that linearizes but does not degrade supercoiled plasmids. Metnase has many but not all of the properties of a transposase, including Terminal Inverted Repeat (TIR) sequence-specific DNA binding, DNA looping, paired end complex formation, and cleavage of the 5′ end of a TIR, but it cannot efficiently complete transposition reactions. Interestingly, Metnase suppresses chromosomal translocations. It has been hypothesized that transposase activity would be deleterious in primates because unregulated DNA movement would predispose to malignancy. Metnase may have been selected for in primates because of its DNA repair and translocation suppression activities. Thus, its transposase activities may have been subverted to prevent deleterious DNA movement.     

Retracted: In Bangladesh,overweight individuals have fewer symptoms of depression than nonoverweight individuals

The aim of this study was to examine whether the association between overweight and depression usually found in western societies would also be found in locations where overweight is not stigmatized. A total of 1,271 individuals from rural Bangladesh were randomly selected; the response rate was 76%. Depressive symptoms were measured with the Montgomery‐Åsberg Depression Rating Scale (MADRS). The sum MADRS scores were 13.4 (s.d. = 5.8) and 18.5 (8.1) for overweight vs. nonoverweight (t = 6.6; P < 0.000) men, respectively, and 19.7 (7.8) and 23.2 (7.9) for overweight vs. nonoverweight women, respectively (t = 4.2; P < 0.000). Thus the MADRS score was lower in overweight individuals. After adjusting for sex and age, BMI significantly predicted the MADRS score (β = ?0.3; t = 10.2; P < 0.000). These findings suggest that overweight may be related to fewer depressive symptoms in non western cultures.     

The M4 muscarinic receptor-selective effects on keratinocyte crawling locomotion

We have investigated how the cholinergic system of epidermal keratinocytes (KC) controls migratory function of these cells. Several molecular subtypes of muscarinic acetylcholine receptors (mAChRs) have been detected in KC. Early results suggested that M(4) is the predominant mAChR regulating cell motility. To determine muscarinic effects on lateral migration of KC, we used an agarose gel keratinocyte outgrowth system (AGKOS) which provides for measurements of the response of large cell populations (> 10(4) cells). Muscarine produced a dose-dependent stimulatory effect on cell migration (p < 0.05). This activity was abolished by atropine, which decreased migration distance when given alone. To identify the mAChR subtype(s) mediating these muscarinic effects, we substituted atropine with subtype-selective antagonists. Tropicamide (M(4)-selective) was more effective at decreasing the migration distance than pirenzepine and 4-DAMP at nanomolar concentrations. We then compared lateral migration of KC obtained from M(4) mAChR knockout mice with that of wild-type murine KC, using AGKOS. In the absence of M(4) mAChR, the migration distance of KC was significantly (p < 0.05) decreased. These results indicate that the M(4) mAChR plays a central role in mediating cholinergic control of keratinocyte migration by endogenous acetylcholine produced by these cells.     

Differential changes in human pharyngoesophageal motor excitability induced by swallowing,pharyngeal stimulation,and anesthesia

We investigated the effects of water swallowing, pharyngeal stimulation, and oropharyngeal anesthesia on corticobulbar and craniobulbar projections to human swallowing musculature. Changes in pathway excitability were measured via electromyography from swallowed intraluminal pharyngeal and esophageal electrodes to motor cerebral and trigeminal nerve magnetic stimulation. After both water swallowing and pharyngeal stimulation, pharyngoesophageal corticobulbar excitability increased (swallowing: pharynx = 59 +/- 12%, P < 0.001; esophagus = 45 +/- 20%, P < 0.05; pharyngeal stimulation: pharynx = 76 +/- 19%, P < 0.001; esophagus = 45 +/- 23%, P = 0.05), being early with swallowing but late with stimulation. By comparison, craniobulbar excitability increased early after swallowing but remained unaffected by pharyngeal stimulation. After anesthesia, both corticobulbar (pharynx =-24 +/- 10%, P < 0.05; esophagus = -28 +/- 7%, P < 0.01) and craniobulbar excitability showed a late decrease. Thus swallowing induces transient early facilitation of corticobulbar and craniobulbar projections, whereas electrical stimulation promotes delayed facilitation mainly in cortex. With removal of input, both corticobulbar and craniobulbar projections show delayed inhibition, implying a reduction in motoneuron and/or cortical activity.     

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.